To get better suggestions, I recommend you share the complete recipe as you used it. The information you provided is not sufficient for a trouble-shooting suggestion to be provided.

Keep acrylamide concentration as u used previously....n increase TEMED concentration

Make the resolving gel solution in advance by adding DD water, stock-A, stock-B and 10%SDS. After that when u r ready to set the gel then pick appropriate amount of this solution and add freshly prepared 10% APS sol. and TEMED. Also increase APS and TEMED concentration.

Hope the gel will solidify properly.

I have followed this method.

You should prepare fresh 10% APS and check the buffers if they are old you should make new buffers. TEMED usually lasts long but if you could get a new TEMED solution that would be great.

Increase APS concentration to 20%

Hope it helps

3/25/21

Dear Shubham,

The answers shown above are all good ones. You should check them out.

Another possibility: When you made your acrylamide and bis-acrylamide stock solutions, are you sure they were the correct concentrations? A simple weighing error could have resulted in one or more of these solutions being at too low a concentration. When combined w/ your buffer, the concentrations would have been too low (dilute) for a stiff gel to have formed after polymerization.

Another possibility: Acrylamide gel polymerization is the result of a free radical reaction that occurs after addition of APS & TEMED. Oxygen is a free radical "trap" that can interfere with/inhibit gel polymerization. Whenever I prepared gels, I would combine the acrylamide, bis-acrylamide and electrophoresis buffer in a small, thick-walled side-arm flask, mix the contents, then apply enough of a vacuum to de-gas the solution (i.e., remove O2). (You don't need a full vacuum, just enough for the solution to boil gently and release dissolved gases. It takes ~15-30 seconds.) After this, I added the APS and TEMED, then cast the gel.

I hope this information helps you.

Bill Colonna Ames, Iowa (USA) [email protected]

always prepare fresh APS and try it all the bufferes should be at RT

Increase TEMED, filter acrylamide solution

Check the APS powder stock. If it’s more than two years, remove and change for a new one.

I had same problem and that was the case.